Our working hypothesis is that successful Hb-based blood substitutes may be generate by cross linking and sulfhydryl modification, with fine-tuning achieved by site-directed mutagenesis that is guided by knowledge of the molecular controls of HB function. This hypothesis is support by our recent discovery that the vasoconstrictive activity of cell-free Hb, attributed to NO scavenging, can be controlled by nitrosylation of its thiol groups. Molecular control functions critical to the design of blood substitutes include the ability of modified Hbs to contribute to oxidative toxicity, redox cycling and NO scavenging/donating reactions. We propose to evaluate these critical functions by spectroscopic measurements of the kinetics and equilibria of heme-and sulfhydryl- ligand binding and novel spectroelectrochemical methods for measuring the heme redox potential. These complementary techniques will be used in parallel to quantify differences between normal and modified Hbs. Our innovative spectroelectrochemical methods allow us to directly compare oxidation and ligand-binding curves and will aid in the design of blood substitutes by differentiating between those aspects of function that are responsive to the redox potential of the active-site iron atoms and those aspects governed by steno features. Hb systems selected for study will enable us to separately test the effects of modifications of the heme pocket, the sulfhydryl groups, and the central cavity. Primary attention will be given to defining the range of functional modifications that result from sulfhydryl modification in combination with chemical cross-linking. Our goal is to trace the molecular pathway from specific sites of modifications to their ultimate functional expression in ligand binding equilibria and kinetics that underlie oxidative toxicity, redox cycling and NO scavenging/donating reactions of cell-free hb.